Fitc or tritc conjugated secondary antibodies

Mouse pancreatic tissues were collected, fixed in paraformaldehyde, dehydrated and then embedded in paraffin. Five‐micrometer thick sections were prepared for immunofluorescence staining. Briefly, after deparaffinization and rehydration, sections were blocked in 5% bovine serum albumin, followed by incubation with primary antibodies overnight at 4°C, and then with FITC‐ or TRITC‐conjugated secondary antibodies (Santa Cruz) for 60 minutes at 37°C. The sections were washed with phosphate‐buffered saline–Tween 20 three times, then mounted with DAPI (Abcam) for nuclear staining. For immunohistochemical staining, horseradish peroxidase‐conjugated anti‐rabbit secondary antibodies were used, and the staining was visualized using diaminobenzidine, followed by counterstaining with haematoxylin. The following primary antibodies were used: anti‐amylase (sc‐46657; Santa Cruz), anti‐insulin (sc‐9168; Santa Cruz), anti‐insulin (ab6995; Abcam), anti‐glucagon (sc‐13091; Santa Cruz), anti‐FGF21 (ab64857; Abcam), anti‐syntaxin‐1 (STX‐1) (sc‐12736; Santa Cruz), anti‐SNAP25 (ab41455; Abcam), anti‐VAMP2 (ab3347; Abcam) and anti‐cleaved caspase‐3 (9664; Cell Signaling Technology).

HCT116 cell layers on glass coverslips were fixed for 15 min using 4% paraformaldehyde, permeabilized for 20 min in PBS containing 0.2% Triton X-100, and then blocked for 2 h with PBS containing 1% BSA and 0.5% goat serum at 37°C. The cells were incubated with primary antibody at 4°C overnight. The antibodies mouse anti-human RRM2, rabbit anti-human CREB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After rinsing with PBS, and then probed with FITC- or TRITC- conjugated secondary antibodies (Santa Cruz, CA, USA) for 1 h at 37°C. The nuclei were stained with DAPI (Sigma) for 15 min. The slides were mounted and visualized by a fluorescence microscope (AX70, Olympus, Tokyo, Japan). The images are representative of triplicate independent experiments.